This project will characterize biochemical and pharmacological aspects of acetylcholine (ACh) storage by rat brain synaptic vesicles. The storage system is composed of a proton-pumping ATPase and a separate ACh transporter that is associated with an allosteric receptor site for the inhibitory drug vesamicol. The previously characterized ACh transporter- vesamicol receptor (AChT-VR) complex of the electric ray, surprisingly, is strongly associated with synaptic vesicle proteoglycan. This project will determine how similar the rat brain AChT-VR is to the electric ray AChT-VR. Rat brain synaptic vesicles will be prepared either in free form (that is, a P3 preparation) or inside metabolically active synaptosomes. Active transport of radiolabeled ACh and binding of radio-labeled vesamicol or a more tightly binding analog to these preparations will be studied using filter assays. Other biochemical, bioorganic and immunochemical techniques also will be used as appropriate. The specific aims are to (1) study the mechanism and specific ion dependence of the rat brain ACh active transport system, (2) study the relationship of vesamicol analog binding to transport inhibition, (3) test whether the AChT-VR in rat vesicles is linked to proteoglycan, (4) identify the rat brain AChT-VR by photo-affinity labeling with a tritiated high affinity ACh analog, (5) develop an assay for the AchT-VR inside of rat brain synaptosomes based on binding of a high affinity, radioactive analog of vesamicol, and (6) subject the synaptosomes to different pharmacological manipulations that alter the metabolism and that might affect the properties of the AChT-VR. The long term goal of the project is to understand the complete biochemistry of ACh storage by synaptic vesicles in mammals, whether it is subject to regulation, and whether storage and release are coupled. This might lead to development of a pharmacology that would increase ACh storage in and release from compromised cholinergic terminals. Since many disease and toxic states impact on the cholinergic nervous system, this capability could be of significant clinical benefit.